1. Institute of Biomedical Chemistry
2. Foundation of Perspective Technologies and Novations
3. Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology
4. Joint Institute for High Temperatures of the Russian Academy of Sciences

The phenomenon of knotted electromagnetic field (KEMF) is now actively studied, as such fields are characterized by a nontrivial topology. The research in this field is mainly aimed at technical applications – for instance, the development of efficient communication systems. Until present, however, the influence of KEMF on biological objects (including enzyme systems) was not considered. Herein, we have studied the influence of KEMF on the aggregation and enzymatic activity of a protein with the example of horseradish peroxidase (HRP). The test HRP solution was irradiated in KEMF (the radiation power density was 10⁻¹² W/cm² at 2.3 GHz frequency) for 40 min. After the irradiation, the aggregation of HRP was examined by atomic force microscopy (AFM) at the single-molecule level. The enzymatic activity was monitored by conventional spectrophotometry. It has been demonstrated that an increased aggregation of HRP, adsorbed on the AFM substrate surface, was observed after irradiation of the protein sample in KEMF with low (10⁻¹² W/cm²) radiation power density; at the same time, the enzymatic activity remained unchanged. The results obtained herein can be used in the development of models describing the interaction of enzymes with electromagnetic field. The obtained data can also be of importance considering possible pathological factors that can take place upon the influence of KEMF on biological objects— for instance, changes in hemodynamics due to increased protein aggregation are possible; the functionality of protein complexes can also be affected by aggregation of their protein subunits. These effects should also be taken into account in the development of novel highly sensitive systems for human serological diagnostics of breast cancer, prostate cancer, brain cancer and other oncological pathologies, and for diagnostics of diseases in animals, and crops.

It is known that electromagnetic radiation of various intensity can have different influence on human body. Electromagnetic fields can have various topology, such as transverse and knotted one (knotted electromagnetic field, KEMF)^1,2. The simplest and most common electromagnetic waves are transverse ones, and, to date, their effects have been widely studied in various frequency and intensity ranges. As regards biomedical applications, microwave radiation is interesting in that, depending on its intensity, it is employed in biological research, and in both medical diagnostics and therapy. In this way, upon exposure of biological tissues to high-intensity radiation (~90 W/cm²), their temperature increases to ~90 °С, and denaturation of biological objects is observed. It was shown that, under such conditions, partial loss of functional activity of proteins (for instance, peroxidase) is observed³. At lower radiation intensity (10 μW/cm^2 4), both positive therapeutic effects (which usually take place owing to local heating⁵) and negative effects are observed. Here, it should be noted that studies on the application of non-thermal effects of low-power microwave radiation (10⁻⁵ to 10⁻³ W/cm²) in cancer therapy were recently reported⁶.

Enzyme systems play an important role in various metabolic processes. For this reason, studying the influence of microwave radiation on enzymes is an important task from the viewpoints of both fundamental and applied science. In this way, an effect of enhancement of erythrocyte membrane resistance upon the exposure of the cells to a radiation of a 1.5 μW power was found⁷. Furthermore, in the studies on the interaction of microwave radiation with proteins, an increase in the catalytic activity of alanine aminotransferase was observed⁸.

From a practical point of view, the microwave (from 2 to 4 GHz) frequency range is the most interesting one. A number of devices called radiometers are functioning in this range. These devices allow one to monitor the functional state of a human body in the event of pathologies⁹, to register the microwave radiation upon excitation of aqueous and protein media¹⁰, and also to monitor the functional activity of protein systems by registering changes in the brightness temperature^11,12. The level of background radiation within a typical radiometer bandwidth (~0.1 GHz) at a temperature of 310 К makes up 4 ? 10⁻¹³ W¹³, while pathologies in human body are accompanied by radiation in the same frequency range at a level corresponding to a change in the brightness temperature of the order of 0.4 °С¹³. In this connection, studies on the influence of electromagnetic radiation in these low-level ranges of power and frequencies on biological objects seem to be relevant.

At present, the theory considering electromagnetic fields with a different topology, known as «knotted electromagnetic fields» (KEMF), is developed; corresponding practical studies were reported^14,15,16,17. These fields have a certain practical relevance, as they can be used in the industry of efficient new generation communication systems. These fields have a specific topology and can influence surrounding biological environment (including staff) — for instance, through the mechanism of changing the physicochemical properties of proteins (particularly enzymes). To date, however, this mechanism has not been studied.

For this reason, in our present work, we have studied the influence of KEMF with 2.3 GHz frequency and a power density of 10⁻¹²W/cm² on an enzyme protein (with the example of horseradish peroxidase, HRP). The KEMF frequency and power have been selected based on the above-listed considerations: such parameters of the electromagnetic field are interesting from the practical point of view for monitoring pathologies in human and for studying enzymatic reactions. HRP protein is a well-studied enzyme and is widely used as a model object in studies of a wide class of peroxidases; this is why this protein has been used in our research. HRP pertains to heme-containing enzymes. Studying peroxidases is of great interest, since these enzymes are well represented in plant and animal tissues¹⁸ and play an important functional role in the organism. Peroxidase catalyzes the oxidation of a broad spectrum of organic and inorganic compounds by hydrogen peroxide¹⁹. For instance, myeloperoxidase plays an important role in human body by participating in atherogenesis²⁰. The molecular weight of HRP is 40 kDa²¹. It is known that the molecules of many enzymes (including HRP) form aggregates²². Changes in the aggregation state of an enzyme under external physicochemical influence (such as thermal, chemical, electromagnetic etc.) characterize a change in its spatial structure. This can lead to pathologies in the organism. It is to be noted that, if the change in the structure does not affect the active site or chromophore groups of the enzyme, it is difficult to reveal such a change by monitoring the kinetic parameters of the catalysis reaction. For this reason, in our present research, atomic force microscopy (AFM) has been employed for studying the influence of electromagnetic field on the HRP aggregation. AFM allows one to perform visualization of protein aggregation and to measure the enzymatic activity at the level of single molecules of enzymes^23,24. AFM has been used to determine the aggregation state of HRP before and after its exposure to KEMF. That is, the enzyme and its aggregates have been visualized on the surface of a solid substrate. In parallel, the enzymatic activity of HRP in solution has been determined by spectrophotometry.

It has been demonstrated that, after the exposure of HRP solution to KEMF, an increased aggregation of HRP macromolecules, adsorbed on the AFM substrate surface, is observed, while the functional activity of the protein remains unchanged. The results obtained herein can be used in the development of models describing the interaction of an electromagnetic field with either isolated enzyme systems or even whole organisms. Moreover, the obtained data are also important for further studies on the development of standards for working with electromagnetic radiation. Furthermore, since protein aggregation can occur in biosensor systems operating in the presence of external electromagnetic fields, the results obtained herein should be taken into account in the development of highly sensitive biosensors (which are sensitive to electromagnetic interference) – including nanowire-based biosensors intended for diagnostics of oncological diseases, such as breast cancer, prostate cancer, brain cancer etc.

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Ivanov, Y.D., Pleshakova, T.O., Shumov, I.D. et al. AFM Imaging of Protein Aggregation in Studying the Impact of Knotted Electromagnetic Field on A Peroxidase. Sci Rep 10, 9022 (2020). https://doi.org/10.1038/s41598-020-65888-z

Nature, Scientific Reports